Nitric Oxide Donor-Based Cancer Therapy - American Chemical Society

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Nitric Oxide Donor-Based Cancer Therapy: Advances and Prospects Zhangjian Huang,†,§ Junjie Fu,‡,§ and Yihua Zhang*,† †

State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China Pharmaceutical University, Nanjing 210009, P. R. China ‡ Department of Medicinal Chemistry, School of Pharmacy, Nanjing Medical University, Nanjing 211166, P.R. China ABSTRACT: The increasing understanding of the role of nitric oxide (NO) in cancer biology has generated significant progress in the use of NO donor-based therapy to fight cancer. These advances strongly suggest the potential adoption of NO donor-based therapy in clinical practice, and this has been supported by several clinical studies in the past decade. In this review, we first highlight several types of important NO donors, including recently developed NO donors bearing a dinitroazetidine skeleton, represented by RRx-001, with potential utility in cancer therapy. Special emphasis is then given to the combination of NO donor(s) with other therapies to achieve synergy and to the hybridization of NO donor(s) with an anticancer drug/agent/fragment to enhance the activity or specificity or to reduce toxicity. In addition, we briefly describe inducible NO synthase gene therapy and nanotechnology, which have recently entered the field of NO donor therapy. generation of peroxynitrite (ONOO−), which further yields powerful oxidants during degradation. (ii) The reaction of NO with O2 (auto-oxidation) or ONOO− produces nitrogen dioxide (NO2), which may result in nitration of tyrosine residues in proteins with an accompanying change in their function.11,12 Therefore, ONOO− can be considered to function as an NO2 donor or an intermediate in the conversion of NO to NO2 via O2•−. (iii) NO2 further reacts with NO to form dinitrogen trioxide (N2O3), which leads to the Snitrosation of thiol-containing proteins via NO+ transferring.11,13 The production of S-nitrosothiol is a post-translational modification (PTM) process that has become increasingly prominent, competing with known PTMs, such as phosphorylation and ubiquitination. It is worth noting that, in addition to concentration, other factors such as the duration of NO exposure and the kinetic behaviors of NO are also key determinants in NO’s biological signaling. Several excellent reviews for detailed information are now available.5,14 1.3. Effects of NO on Cancer Biology. NO has broad effects on cancer, from cancer initiation of cellular transformation to cancer progression of the metastatic cascade. Several recent reviews have described the role of NO in cancer.15−17 NO participates in various signaling pathways, including Ras, extracellular signal-regulated kinases (ERKs), Akt, cyclin D1/retinoblastoma (Rb), and mammalian target of rapamycin (mTOR), which are crucial for tumor cells. Numerous investigations have shown that low levels of NO promote cancer growth by stimulating cancer cell progression and by enhancing angiogenesis and metastasis, while higher

1. INTRODUCTION 1.1. Nitric Oxide and Nitric Oxide Synthases. As an important signaling molecule or effector, nitric oxide (NO) plays an important role in various biological systems.1−5 NO is synthesized in vivo by three different NO synthases (NOSs), among which nNOS and eNOS are constitutively expressed in neuronal and endothelial cells, respectively, and iNOS is regulated transcriptionally and induced by inflammatory cytokines, oxidative stress, aging hypoxia, and various endotoxins.6−8 NOS catalytically converts L-arginine and O2 into NO and L-citrulline, depending on the availability of several cofactors and cosubstrates, such as nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and tetrahydrobiopterin (BH4). NOS isoforms produce different levels of NO, varying from nanomolar (nM) to micromolar (μM), with nNOS and eNOS producing lower basal levels and iNOS generating transient higher concentrations (μM).4,9,10 1.2. Chemical Biology of NO. The biological effects of NO are divided into direct and indirect effects, which highly rely on the concentration of NO over a 1000-fold concentration range.4 Low levels of NO produced by nNOS and eNOS directly interact with specific molecules, such as metals, lipid radicals, and DNA radicals. For instance, the interaction of NO with the iron moiety of soluble guanylyl cyclase (sGC) occurs at nanomolar levels. In contrast, high levels of NO generated by iNOS reacts with various reactive oxygen species (ROS) to produce reactive nitrogen species (RNS), which exert indirect effects, such as oxidation, nitration, and nitrosation. The biological signaling involved in NO’s indirect effects is very complicated, including but is not exclusive to the following examples: (i) The reaction of NO with superoxide anion (O2•−) at equal levels leads to the © 2017 American Chemical Society

Received: November 13, 2016 Published: May 15, 2017 7617

DOI: 10.1021/acs.jmedchem.6b01672 J. Med. Chem. 2017, 60, 7617−7635

Journal of Medicinal Chemistry

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concentrations of NO depress cancer progression by inducing apoptosis, sensitizing tumors to chemo-, radio-, or immunotherapy, reversing resistance to chemotherapy, and retarding the angiogenic and metastatic cascades.18,19 The Wink group examined the effects of five distinct concentrations of NO on MCF-7 cells.4 Low levels of NO target cGMP (1−30 nM), phosphorylate Akt (30−100 nM) and stabilize hypoxia-inducible factor 1α (HIF-1α) (100−300 nM), favoring progrowth and antiapoptotic responses. When the NO levels are high enough to induce p53 phosphorylation (>400 nM) and nitrosative stress (>1 μM), cytostatic and apoptotic responses were observed. Although the NO levels to exert these effects may vary among different cell lines, it nicely exemplified how the bimodal behavior of NO in specific cancer cells could be accurately quantified by its concentrations. High levels of NO may exert their anticancer activity through various mechanisms, including (i) apoptosis stimulation by upregulation of p53;20 (ii) antiapoptotic proteasomal molecule degradation;21 (iii) cytochrome C release and mitochondrial permeability increase;22 (iv) Smac/DIABLO release;23 and (v) peroxinitrite or ONOO− formation, leading to p53 increase,15 cell cycle arrest,24 cell necrosis,25 angiogenesis inhibition,26 and cytotoxicity.27 Bonavidaet et al. proposed that direct NF-κB (p50 and p65) S-nitrosylation, the metastasis inducer Snail, a zinc-finger transcription factor, and the transcription factor Yin Yang 1 (YY1) are involved in the inhibitory mechanism of NO on tumor cell resistance and metastasis. 28 Other in vivo mechanisms have been reviewed eleswhere.29 More recently, Scicinski and co-workers proposed an epigenetic mechanism through which NO oxidation of critical cysteine residues can inhibit histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), which results in multiple biological effects, such as the promotion of p53 expression.30 The role of NO in cancer therapy can also be described from the perspective of the hypoxia and hyponitroxia axis. Hypoxia, a widely accepted hallmark of solid tumors, leads to hyponitroxia by inhibiting NO synthesis, and hyponitroxia heightens hypoxia due to the lack of NO-modulated blood flow.30 The mutually reinforcing cycle of hypoxia/hyponitroxia promotes tumor progression, and either O2 or NO can be targeted for cancer therapy. Since the attempted restoration of O2 in tumor has been largely unsuccessful, NO becomes the relatively operable target. The persistently low NO levels (hyponitroxia) induced by hypoxia in cancer are an optimal “zone” for cancer promotion. If NO levels are shifted even slightly up or down, the “safe zone” becomes a “kill zone” or “inhibition zone” for cancer.30 This provides a therapeutic opportunity to target cancer either by lowering the concentration of NO using NOS inhibitors to inhibit tumor growth or by increasing the level of NO using NO donor-based therapy to induce tumor cell death (Figure 1). A large quantity of studies that shift intratumoral NO above the “safe zone” have been conducted and have yielded significant advances in NO donor-based therapy, including (i) NO donor alone; (ii) combination of NO donor(s) with other therapeutics, such as chemo-, radio-, or immunotherapy to achieve synergistic potency; (iii) hybridization of NO donor(s) with an anticancer drug/agent/fragment to enhance activity or specificity or to reduce toxicity; (iv) iNOS gene therapy to endogenously generate high levels of NO; and (v) utilization of nanotechnology to deliver high levels of NO to the cancer site.

Figure 1. Tumors’ response to different concentrations of NO, which provides a therapeutic opportunity for cancer. Tumors require an optimal concentration of NO for proliferation (safe zone). Very low intratumoral levels of NO inhibit tumor growth (inhibition zone), and very high levels of NO induce tumor cell death (kill zone).

In the present review, we describe the achievements in NO donor-based therapy with a focus on the advances made in the past decade.

2. THERAPEUTIC APPLICATIONS OF NO DONOR-BASED THERAPY 2.1. From NO to NO Donor Compounds. The direct use of NO inhalation for the clinical treatment of persistent pulmonary hypertension of newborn (PPHN) was initiated many years ago and is still on going.31 However, due to the high reactivity and inconvenient handling of this gaseous species, NO donors have gained increasing interest. NO donors are compounds capable of generating NO. Glyceryl trinitrate (GTN) (Figure 2), a well-known organic nitrate that has been clinically used for 150 years to relieve acute attacks of angina pectoris, was not recognized as a NO donor until the 1980s. Other organic nitrates able to release NO include isosorbide dinitrate, isosorbide mononitrate, nicorandil, etc. Several biotransformations of organic nitrates into NO using enzymes have been proposed, including glutathione-S-transferases (GSTs), cytochrome P450 reductase, xanthine oxidoreductase, and aldehyde dehydrogenase-2 (ALDH2).32 The catalysis of ALDH2 has been verified using mutants in vitro and transgenic mice lacking mtADH genes in vivo.33,34 In addition to organic nitrites, many compounds with various structures can generate NO in vitro or in vivo, and each class of compounds generates NO through a different mechanism, e.g., enzymatic, nonenzymatic, and reductive/oxidative. The chemical activities and biological applications of major classes of NO donors, including the organic nitrates, diazeniumdiolates, metal−NO complexes, furoxans, S-nitrosothiols, and sydnonimines (Figure 2), have been comprehensively reviewed by Wang,35 Megson,36 and Bonavida.29 Organic nitrates have recently joined the clinical field of cancer therapy. Organic nitrates affect the tumor vasculature by improving tumor oxygenation and enhancing tumor perfusion.37 GTN and some other nitrates alone or in combination with an anticancer drug exerted significant anticancer activity (see below). Preclinical and clinical data on the anticancer properties of GTN are summarized and discussed elsewhere.38 Glycidyl nitrate (GLYN) (Figure 2) triggered NO release in the M21 melanoma cell line, sensitized SCC VII xenografts to γ irradiation and cisplatin, and increased blood flow in the tumor, which makes it particularly beneficial in tumors with significant 7618



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Figure 2. Structures of some important NO donors.

preparation of new NO donor hybrids, which have been reviewed elsewhere.47,48 Nitrosothiols (RSNOs, one representative is S-nitroso-Nacetylpenicillamine (SNAP)) and sydnonimine (one representative is 3morpholinosydnonimine (SIN1, 1)) (Figure 2) are also important NO donors that have been investigated for therapeutic potential in cancers and have been reviewed elsewhere.29,35 1-Bromoacetyl-3,3-dinitroazetidine (RRx-001, 2) (Figure 2), originally derived from energetic materials, was first synthesized by ATK Aerospace Systems in the U.S.49 Compound 2 exhibited potent antiproliferative activity against a variety of cell lines in different oxygen concentrations in vitro and against multiple tumor types in vivo. Moreover, toxicity studies indicated that 2 was not myelosuppressive, and no doselimiting toxicity was observed following daily administration for 14 days in mice.50 Importantly, 2 acted as a potent radiosensitizer, and its synergistic effects with radiation were intensively studied in different models and reviewed by Oronsky.51 Recently, studies on the metabolism of 2 have confirmed its role as a novel NO donor with unique mechanism of action (Figure 3).52−54 Upon intravenous infusion in rats, approx-

hypoxic areas. In addition, GLYN showed good safety at an effective dose of up to 150 mg/kg in mice.39 N-Diazeniumdiolates (Figure 2) are composed of a diazeniumdiolate [N(O)NO−] moiety bound to a nucleophile amine, including a primary or secondary amine or polyamine, via a nitrogen atom. The salts (usually sodium salts) of diazeniumdiolates are relatively stable in the form of a dry solid but can spontaneously liberate two molecules of NO at physiological conditions following first-order kinetics with a range of half-lives, from a few seconds to several hours.40 Furthermore, the O2-protection (see below) of diazeniumdiolates generates stable neutral prodrugs that can be enzymatically cleaved in tumor cells in a controllable decomposition rate41,42 to produce diazeniumdiolate anion and subsequently release NO spontaneously in situ, exhibiting selective and potent antiproliferative activity. As recently documented by Bonavida and Baritaki, the diazeniumdiolate DETA/NO (Figure 2) exerted anticancer activity by preventing or reversing drug resistance, inhibiting the epithelial−mesenchymal transition, and interfering with the NF-κB/Snail/YY1/RKIP cascade.28 Metal nitrosyl complexes have also been explored as NO donors. Sodium nitroprusside (SNP) (Figure 2), a representative iron−NO complex, has shown therapeutic potential in a series of cancer models through invasion suppression, HIF-1α interference, and radiosensitization.29 It was recently reported that SNP sensitized AGS, SGC7901, MKN45, and MKN28 gastric cancer cell lines to tumor necrosis factor (TNF) related apoptosisinducing ligand (TRAIL) induced apoptosis.43 However, the in vivo effects of SNP were not investigated due to the spontaneous and nonselective NO release. NO-coordinated ruthenium complexes constitute another class of NO donors with the general structure trans-[RuII(NO+)(L1)x(L2)]n+, which can be thermally or photochemically activated to release NO. Compared with other metal nitrosyl compounds, ruthenium nitrosyl complexes have several unique properties, such as high water solubility, stability toward air oxidation, and low cytotoxicity against host cells. The cytotoxicity of some ruthenium nitrosyl complexes against cancer cells has been shown in several studies. For example, trans-[RuII(NO+)(NH3)4(Py)]3+ induced apoptosis in HepG2 cells by decreasing membrane potential and ATP levels, generating ROS, and lowering mitochondrial permeability transition.44 More details are described in the recent reviews by Franco45 and Van de Voorde.46 1,2,5-Oxadiazole N-oxides (furoxans) (Figure 2) are a class of NO donors that release NO in response to thiol-containing molecules, thus generating potent anticancer activity. Furoxans or benzofuroxans are used as synthetic precursors in the

Figure 3. Mechanism underlying NO release by compound 2.

imately 26% of 2 rapidly and irreversibly bound to thiolscontaining electrophiles, such as glutathione (GSH) and cysteine, via the bromoacetamide group to produce soluble GSH/Cys adductive metabolites, which were mainly excreted in the urine in the first 8 h.53 Binding to GSH resulted in depletion of GSH and its precursors in red blood cells (RBCs), increasing oxidative stress and contributing to anticancer activity. Importantly, the rest of infused 2 could permeate into RBCs and rapidly and irreversibly bound to the β-Cys93 7619



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Figure 4. Anticancer drugs used in combination with NO donors.

and 8)/cisplatin (80 mg/m2 on day 1) (Figure 4) led to an improved time to progress (TTP) value compared with vinorelbine/cisplatin alone (327 vs 185 days).61 Similar results were reported in 2014 by Reinmuth et al.62 and Arrieta et al.63 from phase II studies conducted in Germany and Mexico, respectively. The chemo- and radiosensitizing effects of GTN have been confirmed by investigations involving colon cancer, prostate cancer, and rectal cancer.29 In a recent small-scale phase I study, Illum et al. evaluated the maximum tolerated dose of topical GTN in combination with 5-FU (Figure 4) (225 mg/m2 per day) and radiation therapy against advanced operable rectal cancer. No severe toxicity was observed when GTN transdermal patches were applied at a maximum dose of 0.6 mg/h, indicating its good tolerance and safety in clinical use.64 Diazeniumdiolates are also widely used in combination with cytotoxic drugs/agents to achieve synergistic effects in cancer therapy. Concurrent intravenous infusion of PROLI/NO (10−6 M) and carboplatin (20 mg/kg) (Figure 4) resulted in a 40% increase in survival in rats with C-6 gliomas compared with carboplatin or PROLI/NO alone, suggesting that intravenous infusion of PROLI/NO may increase blood−brain barrier (BBB) permeability.65 The combination of DETA/NO (25− 100 μM) with the fanesyltransferase inhibitor SCH56582 (3, Figure 4) at 25 μM led to selective apoptosis of breast cancer cells, accompanied by reduced cytotoxicity on normal breast epithelial cells.66 The chemosensitizing ability of DETA/NO to reverse hypoxia-mediated resistance to chemotherapeutic drugs, such as 5-FU, doxorubicin, cisplatin, and fludarabine (Figure 4), in many types of cancers was observed, including breast cancer, melanoma, neck squamous cancer, and chronic lymphocytic leukemia.29,67 In addition, the immune-sensitization function of DETA/NO has been demonstrated. DETA/NO at 1 mM sensitized prostate cancer cell lines to TRAIL- and FasLmediated apoptosis through NF-κB inhibition via the Snitrosation of p50. The combination of DETA/NO with TRAIL activated the mitochondrial pathway, as indicated by upregulated caspases 9 and 3.68 Other NO donors acting as potential chemo-, radio-, or immunosensitizers include SNP, SNAP, and 2 (see above). For instance, SNP sensitized human gastric cancer cells to TRAIL-induced apoptosis.43

residue of hemoglobin (Hb), which endows 2 with an extended half-life of days to months.53 The binding of 2 with β-Cys93 mimics a ligand−receptor interaction, significantly enhancing the pseudoenzymatic ability of Hb to convert NO2− to NO under profoundly hypoxic conditions in tumors.52 This in situ generated NO under hypoxia could react with ROS to generate ONOO−, N2O3, and other reactive and toxic nitrogen oxides, inducing DNA damage, ATP depletion, apoptosis, and necrosis. In addition, these RNSs could epigenetically modulate DNA methylation, histone deacetylation, and lysine demethylation, leading to p53 reactivation.50,54 As a result, 2 indirectly and locally delivered NO to the tumor site without systemic toxicity, such as hypotension, headaches, and methemoglobinemia, in preclinical toxicology studies. The phase I clinical trial of 2 demonstrated its good activity and tolerance without clinically significant toxic effects at the tested doses.55 On the basis of the above studies, 2 has moved forward to phase II. Pretreatment of 2 for 4 cycles sensitized or resensitized not only refractory small-cell lung cancer (SCLC) patients but also EGFR-inhibitor-resistance and T790Mnegative nonsquamous cell lung cancer patients to the firstline chemotherapeutic drug carboplatin.56,57 2.2. Combination of NO Donors with Other Anticancer Therapies. The application of NO donors in combination with other anticancer therapies has been strongly supported by numerous preclinical and clinical studies. Increased anticancer activity was observed when NO donors were coadministered with conventional chemotherapeutic drugs due to their synergistic anticancer activity. NO donors usually act as sensitizing agents against chemo- or radioresistant cancer cells, which are commonly characterized by hypoxia and the expression of high levels of HIF-1α. Accordingly, NO donors reduce the stabilization of HIF-1α in cancer cells, leading to increased tumor blood flow and oxygenation.58,59 GTN has been clinically demonstrated as a promising chemosensitizing agent, and the most commonly performed clinical trials of GTN are against advanced non-small-cell lung cancers (NSCLCs), which are usually associated with poor chemotherapy outcomes due to tumor hypoxia.60 In a randomized phase II clinical trial in 120 Asian NSCLC patients at stage IIIB/IV in 2006, transdermally applied GTN (25 mg/ patient daily for 5 days) plus vinorelbine (25 mg/m2 on days 1 7620



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FU hybrids.74 However, stability of the O2-(carbonyloxymethyl) moiety in blood plasma remains an innegligible issue. 2.3.1.2. O2-2,4-(Dinitrobenzene) Diazeniumdiolates Activatable by GSH/GST. GSTs are a superfamily of enzymes that promote the nucleophilic conjugation of the thiol group of GSH with an electrophilic xenobiotic, thus increasing its solubility and improving the excretion of the xenobiotic from the cells. GSTs are often overexpressed in cancer cells and are implicated in the development of drug resistance.74 Chlorodinitrobenzene (CDNB), a model substrate used extensively for evaluating the activity of GST, has been reported to conjugate with GSH to form a stable Meisenheimer complex, leading to chloride elimination (Figure 6). Keefer et al. replaced the chloride in CDNB with a diazeniumdiolate moiety to obtain a new class of O2-dinitrobenzene diazeniumdiolate derivatives.75−77 One representative compound, O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) (Figure 6), is the most active antiproliferative agent against HL-60 cells, with an IC50 of 0.2−0.5 μM. Administration of JS-K (iv 4 μmol/kg) reduced HL-60 xenografts by approximately 50% without inducing significant hypotension.75 Further mechanism studies revealed that JS-K increased mitochondrial superoxide levels by nitrating MnSOD at the active site in H1073 cells, caused GSH depletion through the arylating property of JS-K, activated the intrinsic apoptotic pathway in NSCLC cells, blocked androgen receptor and WNT-signaling in prostate cancer cells, and inhibited the transcriptional activity of β-catenin, a protein that plays an important role in cell−cell adhesion and leukemia proliferation, which in turn reduced the levels of cyclin D. McMurtry et al. found that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting the ability of JS-K to induce cancer cell autophagy, which was further confirmed by electron microscopy. In addition, Western blotting showed that JS-K induced the expression of autophagy marker microtubule light chain 3-II in breast cancer cells. Importantly, no autophagy was observed in normal human mammary epithelial cells.78 The investigation on the mechanisms underlying autophagy was awarded the 2016 Nobel Prize. Thus, a deeper understanding of the role of NO in cancer cell autophagy would be of great significance in future research. Further enzymatic studies showed that the reaction of JS-K with GSH was activated by GST-α at a rate 100 times more efficient than the GST-π isoform. Since GST-π is overexpressed in many tumors and GST-α is also expressed in normal cells, PABA/NO was synthesized by replacing the bulky piperazine moiety with a dimethylamine and introducing a substituent on the 5-position of the benzene ring to make the O2-arylated derivatives activated specifically by GST-π (Figure 7).77,79 PABA/NO was more efficiently metabolized by GST-π than by GST-α and exclusively released a greater amount of cytotoxic NO into tumor cells, thus enhancing the antiproliferative activity against A2780 human ovarian cancer xenografts in SCID mice, with an IC50 comparable to that of cisplatin and without weight loss and renal toxicity. PABA/NO increased the nitration of proteins in a dose- and time-dependent manner in mouse skin fibroblast NIH3T3 (WT) cells. A similar positive correlation between dose and cytotoxicity of PABA/NO was observed in HL-60 cells. However, the ester bond in PABA/ NO renders it susceptible to hydrolysis, forming a phenol compound, which preferentially equilibrates to its phenolate ion, and this anion resists nucleophilic attack by GSH, resulting in diminished NO release and anticancer activity.80

Another important application of NO donors in combinational therapy is coadministration with ROS-inducing drugs, such as cisplatin and doxorubicin. NO donor pretreatment in cancer cells could increase the transcriptional activity of the transcription factors activator protein-1 (AP-1) or facilitate the nuclear translocation of NF-κB, both of which augment the rate of transcription of iNOS.69 This NO-iNOS positive feedback generates toxic levels of NO, which reacted with the superoxide anions produced by ROS inducers to yield various apoptosisinducing reactive nitrogen oxides.70 For example, exogenous application of the NO donor SNAP resulted in improved cisplatin cytotoxicity in ovarian cancer cells.71 2.3. Hybridization of NO Donor(s) with an Anticancer Drug/Agent/Fragment. Given that NO is involved in a myriad of biological processes and displays dichotomous (proand antitumorigenic) effects of NO on cancer cells, the selective and effective delivery of NO to tumors is of great importance and has been investigated using a “hybridization” strategy with considerable progress. The strategy usually exploits the abnormalities existing in tumor cells that exist to a lesser extent or not at all in normal cells, such as high or overexpression of certain enzymes, bioreductive environment, acidic microenvironment, and hypoxia conditions, to design target compounds by hybridization of NO donor(s) with an anticancer drug/agent/fragment, preferably via a cancertargeted linker. 2.3.1. O2-Protected Diazeniumdiolates. As mentioned above, diazeniumdiolates spontaneously release NO under physiological conditions (pH 7.4, 37 °C) with half-lives ranging from a few seconds to several hours. O2-Protection of diazeniumdiolate has frequently been used to enhance their stability and selectivity by anchoring the O2-anion to a fragment that can directly act against cancer cells and be activated by specific enzymes that are highly or overexpressed in cancer cells to remove the protective fragment, generating the free diazeniumdiolate which automatically releases cytotoxic NO in situ against cancer. With reliance on the type of enzymes utilized for activation, different groups of O2-protected diazeniumdiolates have been designed and reviewed by Keefer.41,42 2.3.1.1. O2-(Carbonyloxymethyl) Diazeniumdiolates Activatable by Esterases. In contrast to their corresponding free diazeniumdiolates, O2-(acetoxymethyl) diazeniumdiolates 4 and 5 are stable in pH 7.4 buffer but are rapidly hydrolyzed to form the free diazeniumdiolate and subsequently release quantitative NO when exposed to porcine liver esterase (Figure 5).72 Furthermore, incubation of 4 and 5 in leukemia cells, such

Figure 5. O2-(Carbonyloxymethyl) diazeniumdiolates 4 and 5 and their metabolic pathway by esterase.

as HL-60 and U937, also releases a large amount of NO with a different half-life and potently induces apoptosis of the cells. This strategy has been widely expanded to hybridize anticancer agents with diazeniumdiolates using the O2-(carbonyloxymethyl) protective approach to produce compounds such as diazeniumdiolate/peptide hybrids73 and diazeniumdiolate/57621



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Figure 6. Rational design of JS-K and its mechanism of NO release in the presence of GSH/GST.

ium-1,2-diolates was prepared by our group.83 The most active compound 8 (Figure 7) caused significant DNA damage by releasing N,N-bis(2-TsO ethyl)amine and two molecules of NO after activation by GSH/GST in cancer cells. Compound 8 was more cytotoxic than JS-K against human colon carcinoma HCT116, human ovarian OVCAR5, and murine gliosarcoma 9L-2 cells, suggesting that the strategy can be extended to other O2-protected diazeniumdiolates to improve their anticancer activity. 2.3.1.3. INDQ/Diazeniumdiolates Activatable by DTDiaphorase. DT-diaphorase (DTD) is an obligate two-electron reductase. DTD levels are elevated in a number of tumor types, which enables targeted activation of chemotherapeutic quinones in the tumor with minimized normal tissue toxicity.84 Indolequinone is a known substrate for DTD and has been widely used to couple with DNA alkylating compounds at the C-10 position to produce several DTD-activatable prodrugs.85−87 DTD, along with its cofactors NADPH and FAD, can reduce indolequinone to dihydroxyindole, which has increased electron density on the indole nitrogen, triggering rearrangement to release the substituted moiety at the C-10 position. Chakrapani et al. connected a diazeniumdiolate moiety to the C-10 of the indolequinone, generating DTDactivatable NO prodrugs INDQ/NO and 2-Me INDQ/NO (9) (Figure 8).88 In a standard cell viability assay, INDQ/NO was a potent inhibitor at low micromolar concentrations, more active than analogue 9, against human adenocarcinoma cells (DLD1), urinary bladder cells (T24 cells), and cervical cancer cells (HeLa) cells, which are known to overexpress DT-diaphorase.

Figure 7. Structures of PABA/NO and JS-K analogues 6−8.

Several structural modification studies were conducted to improve the activity and selectivity of JS-K-based diazeniumdiolates for the GST-π isozyme and to achieve a better balance between activity and stability. For example, homopiperazine analogue 6 (Figure 7) exhibited potent antiproliferative activity against NSCLC cells in vitro and in vivo, concomitant with activation of the SAPK/JNK stress pathway and upregulation of its downstream effector ATF3.81 Compound 7 (Figure 7), with nitro-to-cyano substitution, showed increased half-life in the presence of GSH without compromising the compound’s in vivo antitumor activity.82 Recently, a new class of O2-(2,4dinitrophenyl)-1-[N,N-bis(2-substituted ethyl)amino]diazen-1-

Figure 8. DT-diaphorase promoted NO release from INDQ/NO and 2-Me INDQ/NO. 7622



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Figure 9. (A) Prodrugs with 1,6-elimination-based linker systems, where XH is masked as a trigger. (B, C) O2-(p-Substituted benzyl) diazeniumdiolate 10 and BORO/NO are activatable by NTR and H2O2, respectively.

Figure 10. Structures of O2-β-galactosyl diazeniumdiolates (β-Gal-NONOate) as well as O2-sialated- and O2-β-D-N-acetylglucosaminyl diazeniumdiolates 11−13.

2.3.1.4. O2-(p-Substituted benzyl) Diazeniumdiolates Activatable by NTR or H2O2. The site-specific activation of nontoxic prodrugs has recently been proposed to enhance the selectivity of drugs for cancer cells. Generally, the prodrugs comprise three parts, i.e., trigger, linker, and effector. 1,6Elimination-based self-immolative linker systems are often used for tumor-activated prodrug therapy,89 which usually involves a trigger masking the linker, such as aniline, N-hydroxyaniline, or phenol moiety, with the drug (effector) attached to the benzylic position directly or through a carbamate or carbonate linkage (Figure 9A). This concept has been successfully employed for the design of site-specific NO donor hybrids, in which the diazeniumdiolate moiety acts as an effector, exemplified by the following two O2-(p-substituted benzyl) diazeniumdiolates. Chakrapani and co-workers designed and synthesized a new class of O2-(4-nitrobenzyl) diazeniumdiolates 10 (Figure 9B).90 In the presence of nitroreductase (NTR), the nitro group in 10 can be reduced to a hydroxylamine. As a consequence, the electron density on the benzyl groups is substantially increased, and the diazeniumdiolate anion is expelled to release NO via a 1,6-elimination. In vitro assay demonstrated that the antiproliferative activity of 10 against DLD-1 and HeLa cells was

significantly enhanced relative to the parent diazeniumdiolate. Similarly, the arylboronate ester-based diazeniumdiolate BORO/NO is activated by hydrogen peroxide (H2O2) to generate NO (Figure 9C).91 Since H2O2 is one of the most common ROS produced in large amounts in several human tumor cells, the potential antineoplastic application of this type of diazeniumdiolate should be investigated. 2.3.1.5. O2-β-Galactosyl Diazeniumdiolates Activatable by β-Galactosidase. O2-β-Galactosylpyrrolidinyl diazeniumdiolate (β-Gal-NONOate) was synthesized by Wang and colleagues and released NO only when hydrolyzed by β-galactosidase in rat glioma cell line 9L with transformed LacZ gene, leading to its more powerful cytotoxicity than that of the free diazeniumdiolate.92,93 In addition, O2-sialated diazeniumdiolate 11 could be efficiently hydrolyzed by neuraminidase to release NO with a Km of 0.14 mM,94 and O2-β-D-N-acetylglucosaminyl diazeniumdiolates 12 and 13 could selectively release NO in macrophages (Figure 10).95 While some of these enzymes are not tumor-specific, application of a site-specific NO-releasing strategy might be worthwhile for the design of novel anticancer drugs/agents. 7623



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Figure 11. Hybrids from NO donor(s) and cytotoxic drugs.

2.3.2. Hybridization of NO Donor(s) with a Known Cytotoxic Drug. The most straightforward method to utilize the anticancer activity of NO is hybridization of NO donors with a known cytotoxic drug. The design of hybrids from NO donors and nucleoside analogues is among the earliest attempts to assess the anticancer activity of NO hybrids. The Naimi group synthesized a group of 3′- and 5′-nitrooxypyrimidine nucleoside nitrate esters and evaluated their anticancer activity in a variety of cancer cell lines. The cytotoxicity of the target compounds was comparable to that of 5-iodo-2′-deoxyuridine but lower than that of 5-fluoro-2′-deoxyuridine.96 The Cai group synthesized diazeniumdiolate/5-FU hybrids, among which 14 and 15 (Figure 11) showed greater inhibitory activity against HeLa and DU145 cancer cells than 5-FU, indicating that the nucleoside and NO could have synergistic anticancer effects in biological systems.74 Recently, hybridization of NO donor(s) with gemcitabine was reported by Li et al.97 Hybrid 16 (Figure 11) showed stronger antitumor activity than gemcitabine due to the synergistic effects of NO and gemcitabine. Platinum-based anticancer drugs are among the most important groups of chemotherapeutic agents. Gou and colleagues developed complexes containing NO donor and platinum-based moieties to improve the cytotoxicity. Compound 17 (Figure 11) was highly toxic to human HCT-116 and SGC-7901 cells. Synergistic effects via DNA binding and NOdonating from the furoxan moiety were confirmed.98 In another work from the same group, organic nitrate/platinum hybrids were investigated as novel anticancer agents. The cytotoxic effect of hybrid 18 (Figure 11) was comparable to that of

cisplatin against HCT-116, HepG2, and MCF-7 cells. However, 18 was 5-fold less cytotoxic than cisplatin to nontumorigenic human liver cell line LO2, suggesting that 18 may be a promising anticancer candidate with selective cytotoxicity to cancer cells.99 Other examples in this class of hybrids include NO/ tamibarotene,100 NO/thalidomide,101 and NO/abiraterone,102as represented by compounds 19−21, respectively (Figure 11). 2.3.3. Hybridization of NO Donor(s) with an Enzyme Inhibitor. Enzyme inhibitors are an important class of anticancer drugs/agents due to their ability to regulate tumorrelated signaling events through enzyme blocking. The hybridization of apoptosis-inducing NO donor(s) with an enzyme inhibitor endows the parent molecule with enhanced anticancer potency and multitarget capacity. NO itself can act as an inhibitor of some enzymes through S-modification. Exemplified below are four successfully designed NO donor/ enzyme inhibitor hybrids published in the Journal of Medicinal Chemistry. Farnesylthiosalicylic acid (FTS) inhibits cancer cell proliferation by functioning as a Ras protein antagonist. Ling et al. designed furoxan/FTS hybrids and tested their anti-HCC activity. Compounds 22 and 23 (Figure 12) significantly inhibited the proliferation of human hepatocellular carcinoma (HCC) cells, superior to either FTS or the furoxan moiety, suggesting that high levels of NO and the inhibition of Rasinvolved signaling may yield a synergistic anticancer effect.103,104 7624



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Figure 12. Enzyme inhibitors and their hybrids with NO donor(s).

WZ4002 (24) (Figure 12) is a promising irreversible epidermal growth factor receptor (EGFR) inhibitor with outstanding selectivity against the EGFR 790M mutant. A group of hybrids from phenylsulfonylfuroxan and anilinopyrimidine, the key scaffold of 24, were synthesized by Han et al. for intervention of NSCLC. Compound 25 (Figure 12) selectively inhibited EGFR mutant L858R/T790M with an IC50 of 0.047 μM. Additionally, 25 exhibited strong antiproliferative activity against EGFR mutation NSCLC cell lines and substantial growth inhibitory effects in an H1975 xenograft mouse model. The NO released from 25 suppressed NF-κB activation, which might improve the response of EGFR mutation-harboring NSCLC to the EGFR inhibitor.105 Histone deacetylase inhibitors (HDACIs) bearing a hydroxamic acid moiety have been exploited as potential anticancer agents, e.g., the approved drug SAHA (vorinostat). On the basis of the fact that NO also inhibits HDACs through S-nitrosation, Duan et al. designed a novel series of hybrids from furoxan and SAHA. Among the target compounds, 26 (Figure 12) exhibited strong antiproliferative activity against a broad panel of cancer cells in vitro and showed dose-dependent antitumor growth effects in an HEL cell xenograft model in vivo.106

Poly ADP-ribose polymerase 1 (PARP-1) inhibitors, such as olaparib, have been intensively evaluated as promising chemopotentiators in several cancer types. However, the targeted delivery of PARP-1 inhibitors to cancer cells remains an unmet need. Maciag et al. conceived a new method to solve this problem by designing NO donor/PARP-1 inhibitor hybrids. Compounds 27 and 28 (Figure 12) were synthesized by hybridization of O2-arylated diazeniumdiolate with olaparib. Upon activation by GST-π, which is overexpressed in several cancer cells, the hybrid molecules simultaneously generated NO and the PARP-1 inhibitor, leading to DNA damage or cross-linking glutathionylation of proteins. The in vivo anticancer effects of 27 and 28 were confirmed using A549 human lung adenocarcinoma xenograft models, and no significant systemic toxicity was observed.107 Selective human carbonic anhydrase (hCA) inhibitors, such as sulfonamide and sulfamate derivatives, represent another class of compounds with broad pharmacological activity. A wide variety of NO donor/hCA inhibitor hybrids were synthesized and tested for their anticancer activity and were recently reviewed by Carradori et al.108 Another example in this category is the NO donor/saquinavir hybrid (29) (Figure 12).109 7625



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Figure 13. Natural products/derivatives and their hybrids with NO donor(s).

from 33 during blood circulation.113 Further investigation led to the identification of hybrid 34 (Figure 13), which acted as a potential agent for colon cancer intervention through the nitration of tyrosine residues in mitochondrial protein, downregulation of the Wnt/β-catenin pathway, and inhibition of the COX-2 centered signaling loop in colon cancer cells.114 The introduction of a cancer-targeted moiety into the hybrid represents another useful strategy to achieve tumor selectivity. As a proof of concept, Fang et al. synthesized nitrate/OA hybrids with an amino acid/dipeptide moiety as a linker attached to the C28 of OA. The amino acid/dipeptide moiety is utilized to mimic the substrate of peptide transporter 1 (PepT1), which is relatively overexpressed in several cancer cell lines. Two hybrids, 35 and 36 (Figure 13), showed potent cytotoxicity because of their higher PepT1 affinity. Meanwhile, the hydrophilic peptide moiety resulted in improved aqueous solubility of the hybrids.115 Most recently, Fang et al. designed OA/NO/platinum(II) trihybrid molecules. The trihybrid 37 (Figure 13) selectively inhibited HCC cells as a result of the synergistic effects of cytotoxic NO and OA and the DNA-binding activity of Pt(II).116 Synthetic OA derivatives, such as 2-cyano-3,12-dioxooleana1,9(11)-dien-28-oic acid (CDDO), CDDO-Me, CDDO-Im, and olean-28,13β-olide, are promising lead compounds with remarkably enhanced anticancer activity relative to OA. Ai et al. synthesized CDDO-PepT1 targeting linker-NO donor trihybrids to achieve combined effects. The most active compound, 38 (Figure 13), inhibited the proliferation of not only drugsensitive colon cancer cells but also their drug-resistant counterparts. Furthermore, 38 repressed the growth of

2.3.4. Hybridization of NO Donor(s) with an Active Natural Product. Natural products provide an important source of anticancer lead compounds. During the past decade, one of our group’s interests has been to develop novel anticancer agents through hybridization of NO donor(s) with oleanolic acid (OA), a naturally existing pentacyclic triterpenoid with demonstrated inhibitory activity against hepatitis and tumors. The first candidate, 30 (Figure 13), was discovered by Chen et al. from a group of furoxan-based NO donor/OA hybrids. Compound 30 exhibited strong cytotoxicity selectively against human HCC cells and drastically impeded the growth of HCC tumors in vivo.110 However, the low aqueous solubility of 30 was a major challenge for its further clinical application. To overcome this problem, Huang et al. synthesized a series of glycosylated prodrugs of 30, among which the galactosylated derivative 31 (Figure 13) showed encouraging anti-HCC activity, with an approximately 8 times greater aqueous solubility than that of 30.111 Subsequently, O2-protected diazeniumdiolates were used instead of furoxan to construct new NO donor/OA hybrids to further enhance cancer cell selectivity. Compound 32 (Figure 13), bearing an O2glycosylated diazeniumdiolate moiety, inhibited HCC tumor growth in mice at 3 mg/kg (iv) and showed low acute toxicity (LD50 = 173.3 mg/kg).112 Fu et al. designed hybrids from O2(2,4-dinitrophenyl)diazeniumdiolate and OA using various amino acids as linkers. The hybrid 33 (Figure 13), with GST-π selectivity, released high levels of NO in HCC cells, which may contribute to its remarkable antiproliferative activity in vitro and significant tumor-suppressing ability in vivo. Compared with JS-K and PABA/NO (Figures 6 and 7), 33 showed significantly enhanced stability toward GSH in the absence of GST-π, thus avoiding the off-target release of NO 7626



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Figure 14. Hybrids from NO donor(s) and MDR reversal agents.

K562/A02 cells as revealed by the Griess assay, which might account for their potent MDR reversal activity.128 Our group is interested in the development of hybrids from NO donors and dimethyl-4,4′-dimethoxy-5,6,5′,6′dimethylenedioxybiphen-yl-2,2′-dicarboxylate (DDB), which was demonstrated to reverse Pgp-mediated MDR through intracellular anticancer drug accumulation and induction of Pgp inhibition-mediated apoptosis. Tang et al. discovered that hybrids 47 and 48 (Figure 14) significantly reversed the resistance of MCF-7/Adr cells to doxorubicin. After treatment of MCF-7/Adr cells with 47 or 48 in addition to doxorubicin, the intracellular concentration of doxorubicin was markedly increased via down-regulation of Pgp expression and blockage of Pgp-mediated drug efflux. The NO released by 47 and 48 exerted synergistic effects and chemosensitized MCF-7/Adr cells.129 More recently, Gu et al. synthesized compounds 49 and 50, which effectively inhibited the viability of both drugsensitive and MDR tumor cells. They concluded that 49 and 50 induced mitochondrial tyrosine nitration and apoptosis, downregulated HIF-1α expression, and suppressed the activation of AKT, ERK, and NF-κB in MDR cells.130,131 2.3.6. Hybridization of NO Donor(s) with an NSAID. Nonsteroidal anti-inflammatory drugs (NSAIDs) have recently received increased interest due to their potential protective role against cancer. Hybrids of NO donors and NSAIDs (NONSAIDs) have emerged as promising anticancer agents. A large group of NO-NSAIDs with the structure “NSAID-linker-NO donor” was synthesized by Thatcher and co-workers and was

human drug-resistant colon cancer xenografts in mice by ∼60%.117 Other examples of NO donor/natural product hybrids for cancer intervention include NO/glycyrrhetinic acid (GA),118 NO/oridonin,119,120 and NO/matrine,121 represented by compounds 39−41, respectively (Figure 13). 2.3.5. Hybridization of NO Donor(s) with an MDR Reversal Agent. Multidrug resistance (MDR) in cancer cells is a major hurdle to efficient chemotherapy. One of the main mechanisms of MDR originates from overexpressed ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp). Classical NO donors, such as SNAP, SNP, S-nitrosoglutathione (GSNO), and furoxan derivatives, are able to inhibit ABC transporters overexpressed in MDR tumor cells by nitrating critical tyrosine residues. It has been shown that impairment of these transporters reduces the efflux of doxorubicine (DOX) in cancer cells, with a resulting increase in cytotoxicity.122−124 On this basis, Riganti and colleagues synthesized NO-donating DOXs, represented by compounds 42 and 43, which increased DOX accumulation in MDR cancer cells with a resulting increase in cytotoxicity.125−127The potential use of NO donor/ MDR reversal agent hybrids has also been intensively investigated. NO donors are usually linked to Pgp inhibitors or modulators with MDR reversal activity. Zou et al. synthesized a panel of furoxan-based NO donor/tetrahydroisoquinoline (THIQ) hybrids. At a concentration of 10 μM, hybrids 44−46 (Figure 14) produced 9−13 μM nitrites in 7627



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Figure 15. Hybrids (51−55) from NO donor(s) and NSAIDs as well as NOSH-aspirin from NO/H2S donors and aspirin.

conventionally named with the prefix “NCX”.132 For instance, NCX-4016 (51) and NCX-4040 (52) (Figure 15) are NOaspirin hybrids in which the nitrate moiety is connected to the carboxylic acid group of aspirin via a benzyl moiety. NO-aspirin possesses increased antiproliferative and proapoptotic activities compared to aspirin or the NO donor alone,133 and their mechanisms of action include inhibition of cancer cell growth through redox-dependent signaling,134 suppression of NF-κB signaling in estrogen receptor negative breast cancer cells,135 and G2/M phase cell cycle arrest by regulating phase transition proteins.136 Both NO and aspirin are essential for the anticancer activity, and some studies also claimed that the quinonemethide intermediate plays a key role.137 Another successful example of NO-NSAIDs is the hybridization of NO donors with sulindac to yield NO-sulindac, such as NCX-1102 (53) (Figure 15). Stewart et al. demonstrated that 53 exhibited proapoptotic and anti-invasive effects against PC3 prostate cancer cells, superior to sulindac. Treatment with 53 decreased HIF-1α expression and reduced Akt phosphorylation in the cells.138 Other NO-NSAIDs with confirmed anticancer activity include NO-salicylic acid, NO-ibuprofen, and NO-flurbiprofen, which have been reviewed by Rigas and Kashfi.133 The hybridization of furoxan with (S,R)-3-phenyl-4,5dihydro-5-isoxazole acetic acid led to another well-studied NO-NSAID, GIT-27NO (54) (Figure 15).139,140 While the anti-inflammatory parental drug showed no anticancer effects, hybrid 54 acquired powerful anticancer activity, which was confirmed both in vitro and in vivo. The mechanism of action study revealed that 54 activated mitogen-activated protein kinase (MAPK) family members ERK, p38, and Jun N-terminal kinase (JNK). Additionally, 54 triggered different types of programmed cell death (PCD) in various cell lines. In some cell lines, such as the astrocytoma C6 and melanoma B16, no classical apoptotic signs were induced by 54; however, high levels of autophagosomes were observed in the cytoplasm, suggesting that 54 may induce cancer cell death by autophagy in these cell lines.140 In recent years, an even bolder design from the Kashfi group led to the discovery of “super” aspirin, designated NOSHaspirin (Figure 15), where hydrogen sulfide (H2S) donor and NO donor moieties are covalently linked to the 1,2 positions of aspirin. NOSH-aspirin inhibited the growth of colon cancer

HT-29 cells with nanomolar IC50 values (9000 times more potent than the sum activity of aspirin, the NO donor, and H2S donor moieties).141 As claimed by the authors, “this is the first NSAID based agent with such high degree of potency”. In vivo investigation revealed that NOSH-aspirin caused an 85% reduction in the volume of human colon cancer xenografts in mice. Currently, studies aiming to develop NOSH-aspirin analogues and to clarify the structure−activity relationship of these compounds are in progress.142,143 Meanwhile, the “dual donor” strategy has been applied to produce NOSH-sulindac (55) (Figure 15), which inhibited the growth of several human cancer cells at rates 1000−9000 times more potent than sulindac.144 2.4. iNOS Gene Therapy. The role of iNOS in tumor development is very complex, and both carcinogenic and anticarcinogenic effects of iNOS have been reported. Either induction or inhibition of iNOS has anticancer potential based on the tumor response to different concentrations of NO (see above). The hypoxic environment resulting from iNOS inhibition can be utilized to boost the cytotoxicity of bioreductive drugs. However, it might interfere with the potency of traditional chemotherapy and radiotherapy.145 Conversely, iNOS-based suicide gene therapy, which produces elevated NO levels to elicit tumoricidal effects through more specific and localized expression of the iNOS gene, represents a promising strategy. Currently, many types of vectors have been employed for iNOS gene delivery, including adenoviral vectors, cationic liposomes, bioinspired recombinant vectors, and retroviral vectors. The effects of iNOS gene therapy have been demonstrated on various cancer cells, such as ZR-75-1 breast cancer, A549 lung cancer, and T24 bladder carcinoma.146−148 Furthermore, the in vivo anticancer potency was confirmed in numerous animal models, including human medullary thyroid carcinoma-bearing rat, human colon cancer xenografts in mice, colorectal adenocarcinoma xenografts in SCID mice, and the A549 metastasis model.147,149−151 2.5. NO-Donating Nanoparticle Systems. As an alternative to small NO donor molecules, the development of NO-donating nanoparticles as potent tumoricidal agents has recently attracted significant attention due to their unique advantages. First, nanoparticles can strengthen the stability of the NO donor within the nanosystems compared with the free 7628



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with several well-known NO donors. Further investigations of this type of compound will expand the structural scope of NO donors and promote the clinical application of NO donor-based therapy. The combination of an anticancer drug/agent with NO donor(s) continues to be a good choice for NO donor-based therapy. The most important factors are the type, amount, and/ or preparation formulation of NO donor(s) to achieve the best synergistic effects. The hybridization of an anticancer drug/agent/fragment with NO donor(s) via a cancer-targeted linker, by acting in tumors to display synergistic antitumoral effects while sparing healthy cells, may result in hybrids that are more effective and less toxic than the parent drug/agent. However, the real beneficial effects should be observed in both pharmacodynamic and pharmacokinetic studies in vivo. In addition, NO-donating nanoparticle systems, which have recently been applied to the field of NO donor-based therapy, possess great potential to store and site-specifically deliver high levels of NO to the cancer site and deserve intensive investigation. Low constitutive levels of NO induced by hypoxia in tumors are optimal for the mediation of aberrant, proliferative signaling, leading to progression of cancer cells, whereas levels above or below this optimal range can have the opposite effect and activate signal transduction pathways that may cause cell growth inhibition or cell death.30 These studies suggest that it may be essential to ascertain the minimum threshold dose of NO before a switchlike response from promotion to inhibition or killing of a tumor is elicited. In this regard, it should first be precisely determined what levels of NO are optimal for cancer cell promotion and then specifically kill or inhibit tumor cells in vivo using NO donor-based therapy at appropriate doses with the knowledge of the interactions of NO with the signaling proteins in tumor cells. As for the new generation of NO donors, chemical synthesis is not a difficult job from the viewpoint of medicinal chemistry, but for therapeutic uses, these compounds must have tissue selectivity and demonstrate cancer-targeted NO release in a controlled and sustained manner to avoid the drawbacks related to the rapid burst of NO associated with traditional NO donors, such as nitrates. The O2-protected diazeniumdiolates may be preferable to other NO donors since a rationally designed protecting group that can be metabolically removed by enzymes that are highly or overexpressed in cancer tissues or cells would result in relatively slow and long-lasting NO release at the cancer site. Additionally, further studies into new imaging examinations will be required. For instance, NO-donating compounds may be connected to a fluorescent group to monitor and/or capture the in vivo effects of NO on tumors. It is also worth mentioning that in some cases, the anticancer activity of a NO donor cannot be simply attributed to NO release alone. For example, compound 1 is activated by O2 to produce both NO and O2−, leading to ONOO− formation. The anticancer mechanisms of 2 and JS-K involve GSH depletion. The anticancer potency of NO donor hybrids usually results from the synergistic effects of NO and the conjugated moiety. The linker can also act as a tumor-targeting motif or decompose into an active metabolite, such as the quinonemethide generated from compounds 51 and 52. Therefore, there is great flexibility for organic chemists and medicinal chemists to design NO-donating anticancer agents using state-of-the-art methods. Of course, it also poses more challenge for the

NO donor. Second, because of the high surface area, nanoparticles can load large amounts of NO and undergo passive targeting to solid tumors via the well-known phenomenon of the “enhanced permeability and retention” (EPR) effect.152 Third, there is a possibility of triggering NO release from the nanomaterial directly to the tumor site by regulating the light input, temperature, pH, and magnetic field. In this regard, NO-donating nanoparticle systems could localize high concentrations of NO in a sustained manner to the tumor site rather than circulating to other organs, thus avoiding systemic toxicity. Many NO-donating nanoparticle systems to fight against tumor cells have been prepared based on polymeric nanoparticles, dendritic polymers, liposomes, silica nanoparticles, metallic nanoparticles, and quantum dots and have been reviewed.153−156 Of course, each of these systems has pros and cons that should be carefully considered when being utilized. In addition, some NO-donating nanoparticle systems are complicated and contain nonbiocompatible or nonbiodegradable components, which lower the repeatability or druggability. The preparation of a truly biocompatible and biodegradable platform for NO delivery is still under research. Another unique property of NO-donating nanoparticle systems is that it provides an easy and flexible method to combine an NO donor with cytotoxic agents. For example, a hollow microsphere (HM) system carrying the anticancer agents irinotecan and DETA/NO was utilized to treat MDR cancer.157 Upon injection of this system into slightly acidic tumor tissue, environmental protons infiltrate the shell of the HMs and react with their encapsulated DETA/NO to produce NO bubbles that initiate localized drug release, which can serve as a Pgp-mediated MDR reversal agent. The site-specific drug release and the NO-reduced Pgp-mediated transport result in the intracellular accumulation of the drug at a concentration that exceeds the cell-killing threshold, eventually inducing cancer cell apoptosis.

3. CONCLUSION AND OUTLOOK NO donor-based therapy mainly consists of iNOS gene therapy and NO donor(s) alone or in combination/hybridization with an anticancer drug/agent/fragment. While iNOS gene therapy can enhance NO delivery for the intervention of cancers, this strategy must overcome two substantial obstacles before making breakthrough progress. One is the risk from the viral vectors that are required to deliver the gene therapy, and the other is the early death of iNOS transfectants.158,159 In contrast, NO donor(s) alone or in combination/hybridization with an anticancer drug/agent/fragment can induce a multitude of antitumor activities, encompassing induction of apoptosis, sensitization to chemo-, radio-, or immunotherapy, and inhibition of metastasis, angiogenesis, and hypoxia. Accordingly, therapeutic application of these approaches may be considered a promising anticancer strategy. Among the current NO donors, only organic nitrates and SNP are available for clinical use. However, patients taking long-term nitrates often develop tolerance, and prolonged SNP administration may lead to cyanide accumulation in the body. How to address these issues remains a challenging subject for NO donor-based therapy. The recently emerging dinitroazetidine compounds, such as 2, which function essentially as NO donors, generate and selectively deliver high levels of NO under hypoxic conditions at a sustained but accelerated speed to exert potent antitumor activity. At therapeutic doses, these compounds do not show the systemic toxicity that is associated 7629


Journal of Medicinal Chemistry comprehensive mechanism of action studies on these compounds. There is an unmet medical need for the discovery and study of a new generation of NO donor-based therapy with controlled release of appropriate levels of NO at the cancer site to generate a desired response, thereby tipping the balance from cancer cell survival to death. We believe that this innovative NO donor-based cancer therapy may facilitate the clinical development of anticancer drugs.

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ABBREVIATIONS USED

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REFERENCES

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ABC, ATP-binding cassette; ALDH2, aldehyde dehydrogenase2; AP-1, activator protein 1; ATF3, activating transcription factor 3; BBB, blood−brain barrier; BH4, tetrahydrobiopterin; CDNB, chlorodinitrobenzene; cGMP, cyclic guanosine monophosphate; COX-2, cyclooxygenase 2; DNMT, DNA methyltransferase; DTD, DT-diaphorase; EGFR, epidermal growth factor receptor; eNOS, endothelial NO synthase; EPR, enhanced permeability and retention; ERK, extracellular signal-regulated kinase; FAD, flavin adenine dinucleotide; FTS, farnesylthioslicylic acid; GA, glycyrrhetinic acid; GLYN, glycidyl nitrate; GST, glutathione-S-transferase; GTN, glyceryl trinitrate; H2O2, hydrogen peroxide; hCA, human carbonic anhydrase; HCC, hepatocellular carcinoma; HDAC, histone deacetylase; HDACI, histone deacetylase inhibitor; HIF-1α, hypoxia-inducible factor 1α; HM, hollow microsphere; iNOS, inducible NO synthase; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MDR, multidrug resistance; mTOR, mammalian target of rapamycin; N2O3, dinitrogen trioxide; NADPH, nicotinamide adenine dinucleotide phosphate; NF-κB, nuclear factor κ-light-chain-enhancer of activated B cells; NMDA, N-methyl D-aspartate; nNOS, neuronal NO synthase; NO, nitric oxide; NO+, nitrosonium ion; NO2, nitrogen dioxide; NO2−, nitrite anion; NOS, NO synthase; NSAID, nonsteroidal anti-inflammatory drug; NSCLC, nonsmall-cell lung cancer; O2•−, superoxide anion; OA, oleanolic acid; ONOO−, peroxynitrite; PARP-1, poly ADP-ribose polymerase 1; PCD, programmed cell death; PepT1, peptide transporter 1; Pgp, P-glycoprotein; PPHN, persistent pulmonary hypertension of newborn; PTM, post-translational modification; RKIP, Raf kinase inhibitor protein; RNS, reactive nitrogen species; ROS, reactive oxygen species; SAPK, stress activated protein kinase; SCLC, small-cell lung cancer; sGC, soluble guanylyl cyclase; Smac/DIABLO, second mitochondriaderived activator of caspase/direct inhibitor of apoptosisbinding protein with low pI; SNP, sodium nitroprusside; STAT1, signal transducer and activator of transcription 1; TRAIL, tumor necrosis factor related apoptosis-inducing ligand; TTP, time to progress

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 025 83271015. E-mail: [email protected]. ORCID

Junjie Fu: 0000-0002-2741-7469 Yihua Zhang: 0000-0003-2378-7064 Author Contributions §

Z.H. and J.F. contributed equally.

Notes

The authors declare no competing financial interest. Biographies Zhangjian Huang received his B.S. and Ph.D. degrees from China Pharmaceutical University (CPU) in 2004 and 2009, respectively. From 2009 to 2012, he worked as a postdoctoral fellow supervised by Prof. Edward E. Knaus at University of Alberta in Canada. He joined the research group of Prof. Yihua Zhang at CPU in 2013. He is now Professor of Medicinal Chemistry at CPU. His research interests involve the discovery of novel anticancer, anti-inflammation, and cardiovascular-disease-related agents based on gaseous messengers such as nitric oxide (NO), nitroxyl (HNO), hydrogen sulfide (H2S), and some natural products. Junjie Fu obtained his Ph.D. degree in Medicinal Chemistry from China Pharmaceutical University (CPU) under the supervision of Prof. Yihua Zhang and Prof. Sixun Peng in 2013. Afterward, he worked as a postdoctoral fellow in the Department of Medicinal Chemistry at University of Florida until 2015. At present, he is an Associate Professor of Medicinal Chemistry in Nanjing Medical University. His research activity is focused on using rational drug design to identify and develop novel anticancer drugs including NO-donating agents. Yihua Zhang graduated from China Pharmaceutical University (CPU) in 1970. He continued his education in medicinal chemistry under the supervision of Prof. Sixun Peng at CPU in 1979 and received his M.S. degree in 1981. He completed his Ph.D. studies in Organic Chemistry under the direction of Prof. Salo Gronowitz at Lund University in Sweden in 1994. He subsequently joined the group of Prof. T. Y. Shen as a postdoctoral fellow in medicinal chemistry at University of Virginia in the United States. Since 1997, he has been Professor of Medicinal Chemistry working at CPU. His research interests mainly focus on the nitric oxide donating drugs.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (Grants 81273378, 21372261, 81673305, and 81602960) and Jiangsu Province Funds for Distinguished Young Scientists (Grant BK20160033). Part of the work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Program for New Century Excellent Talents in University (Grant NCET-13-1033), Startup Funding for Introduced Talents of Nanjing Medical University (Grant KY109RC1602), and Jiangsu Shuang Chuang team. 7630



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